Recombinant lactobacillus plantarum oral vaccine for preventing and controlling ASFV mucosal infection as well as preparation method and application of recombinant lactobacillus plantarum oral vaccine

Abstract

The invention relates to the technical field of biological genetic engineering, in particular to construction of recombinant lactobacillus plantarum of an African swine fever virus antigen, and the method comprises the following steps: constructing a lactobacillus plantarum shuttle vector containing different ASFV antigen genes; and referring to a genome sequence of a Chinese isolate CADCHN09 of ASFV disclosed in GenBank. The method has the advantages that an inducer is not needed, antigen protein is efficiently expressed, the dendritic cell (DC) binding peptide for enhancing immune cell uptake and processing is subjected to fusion expression, the oral immunization mode is simple, mucosal immunity is stimulated, and ASFV infection is effectively prevented and controlled; probiotics, namely lactobacillus plantarum, which naturally exist in animal intestinal tracts and can be colonized and copied in digestive tracts for a long time are used as a carrier; key epitopes of proteins such as nucleocapsid protein p72, outer envelope protein CD2v, inner envelope protein p54, p22, pE199L and pE248R, which play an important role in adsorption, internalization, endocytosis and membrane fusion of ASFV, are preferably fused with dendritic cell binding peptide and then are displayed on the surface of lactobacillus plantarum.

Description

Technical Field

[0001] The present invention relates to the technical field of biological genetic engineering, and specifically to a recombinant Lactobacillus plantarum oral vaccine for preventing and controlling ASFV mucosal infection, its preparation method and application. Background Art

[0002] African swine fever is an acute, febrile, highly contagious disease of pigs caused by the infection of African swine fever virus. The disease is characterized by high fever, anorexia, bleeding of the skin and internal organs in clinically affected pigs, and a high mortality rate. The disease poses a great threat to the global pig industry and is listed as a class I animal disease in the list of animal pathogenic microorganisms in China. Since African swine fever was introduced into China in 2018, it has caused a devastating blow to the pig industry in China. Although the spread and harm of African swine fever epidemic in China have been alleviated due to the general strengthening of biosecurity measures in the pig industry in China, it is still the number one disease threatening the pig industry in China. The pig industries in all countries in the world are looking forward to an efficient vaccine for preventing and controlling African swine fever. However, due to the complex structure of African swine fever virus, the vaccines developed by traditional methods have poor effects.

[0003] ASFV is the only member of the genus African swine fever virus in the family African swine fever virus. The viral genome is double-stranded DNA, with a length ranging from 170 kb to 193 kb, encoding 150-167 proteins. The virus particles of ASFV have an icosahedral structure and are divided into five layers: core, inner capsid, inner membrane, outer capsid, and outer membrane. Currently, 19 genes are known to encode structural proteins. The inner core shell is composed of polyproteins such as pp220 and pp62. The inner membrane is mainly derived from the endoplasmic reticulum membrane and contains p54, p17, and p12 proteins. The capsid is composed of more than 2,000 hexagonal capsomeres, mainly p72 protein, accounting for 33% of the total virus particles. The envelope is obtained from the plasma membrane of the cell when ASFV buds. Due to the large genome of ASFV and the numerous proteins encoded by the virus, the functions of quite a number of viral proteins and the antigen genes inducing protective immunity are still unclear; moreover, the virus particles have a multi-layered complex structure and have a strong ability to evade the host immune system. Porcine primary alveolar macrophages (PAMs) are the main target cells of ASFV. Except for host monocytes / macrophages, ASFV is difficult to continuously grow and reproduce in other in vitro cell lines. Therefore, the in vitro culture of ASFV requires the sacrifice of healthy live pigs. In addition, as a highly pathogenic pathogen, the culture of ASFV needs to be carried out in a biosafety level 3 laboratory, and the virus culture cost is extremely high.

[0004] ASFV can be transmitted by direct contact with infected animals or by the bites of soft ticks infected with the virus. It can also be transmitted directly or indirectly through contact with the excretions or secretions of infected pigs, such as feces, blood, urine, saliva, etc., and through contact with contaminants such as feed, equipment, clothing, shoes, and transportation tools contaminated with the virus. In addition, ASFV can also be transmitted by short-distance aerosol. Therefore, developing a vaccine that can induce a mucosal immune response against ASFV will have broad application prospects.

[0005] Currently, the R & D strategies for African swine fever vaccines at home and abroad mainly focus on three aspects: one is to use gene editing technology to knock out the known virulence genes of ASFV to make attenuated live vaccines or isolate natural attenuated strains from nature. However, current research results show that there is a high risk of homologous recombination between the attenuated live vaccine and the wild virus prevalent in the pig population, resulting in the reversion of virulence. The attenuated live vaccine often causes varying degrees of side reactions after use. Another is to prepare subunit vaccines by expressing ASFV proteins. Subunit vaccines do not contain viral nucleic acids and cannot replicate autonomously in the body, and their safety is significantly higher than that of attenuated live vaccines. However, the immune response induced by the currently developed subunit vaccines used by intramuscular injection is mainly humoral immunity, and the induced antibodies mainly exist in the blood. Such antibodies can only directly contact the virus to play a role when the virus enters the blood and causes viremia, and cannot block the infection of the virus through the digestive and respiratory mucosa in the first place. The third is to use other viruses as vectors, insert different antigen genes of ASFV into the genome of the vector virus, and use the recombinant virus expressing different antigens as a vaccine for intramuscular injection for immunization. It has been reported at home and abroad that antigen combinations of ASFV are expressed using virus vectors such as adenovirus, pseudorabies virus, poxvirus, and vaccinia virus. The results show that there are great differences in the immune protection provided by different antigen combinations and vectors. It is indicated that the antigen combination and the vector delivering the antigen are also particularly important. Previous studies have shown that antibodies against the p72 and p54 proteins of ASFV can inhibit virus adsorption, and antibodies against the p30 protein can inhibit the internalization of ASFV by cells; the recombinant p72, p54, and p30 proteins prepared alone or in combination as vaccines can induce the production of neutralizing antibodies in pigs through the intramuscular injection route, but cannot provide protection against ASFV infection, indicating that the antigen combination, the immune route of the vaccine, and the vector delivering the antigen are all crucial for obtaining an ideal immune protection effect.

[0006] In China, Saccharomyces cerevisiae has been used as a vector for surface display to express different antigens of ASFV, and then different recombinant yeasts are mixed in a certain proportion to prepare an oral vaccine for preventing and controlling ASFV. After immunization and challenge, almost no virus is excreted from mucosal tissues such as the oral cavity, nasal cavity, and anus; immunized pigs can resist oral infection with a certain lethal dose of ASFV. This case shows that mucosal immunity is crucial for preventing and controlling ASFV infection. However, Saccharomyces cerevisiae is not a natural inhabitant of the animal body's digestive tract, and its colonization and reproduction ability in the intestine is very limited. During use, it needs to be repeatedly used, and it cannot achieve the effect of providing long-term immunity with a single use, which is urgently needed in the pig industry. In addition, the Saccharomyces cerevisiae expression system requires the addition of expensive inducers during cultivation, and some inducers are also toxic to animals. More importantly, this recombinant strain that requires the addition of inducers for in vitro culture cannot use inducers after being fed to animals and entering the digestive tract. The recombinant engineering bacteria that enter the body cannot continuously express ASFV antigens in the absence of inducers, which will greatly affect the effect of the oral vaccine. Summary of the Invention

[0007] The purpose of the present invention is to provide a recombinant Lactobacillus plantarum oral vaccine for preventing and controlling mucosal infection of ASFV, its preparation method and application. The present invention has the advantages of not requiring inducers, efficiently expressing antigen proteins, fusion expression enhancing immune cell uptake and processing, simple oral immunization method, stimulating mucosal immunity, and effectively preventing and controlling ASFV infection, and solves the problems raised in the above background technology.

[0008] To achieve the above purpose, the present invention provides the following technical solutions: Construction of recombinant Lactobacillus plantarum of African swine fever virus antigen, and its method includes the following steps:

[0009] S1: Construction of a Lactobacillus plantarum shuttle vector containing different antigen genes of ASFV.

[0010] S1.1: Refer to the genomic sequence of the ASFV Chinese isolate CADC_HN09 published in GenBank to obtain the protein sequences of p72, p54, p49, p32, p22, B602L, CD2v, E120R, E199L, and E248R respectively. Use the Protean program in the Lasergene software package to analyze the antigenic site concentration regions of each protein. For p72, select amino acids at positions 136 - 326 (shown in SEQ ID NO.1), for p54, select amino acids at positions 61 - 184 (shown in SEQ ID NO.2), for p49, select amino acids at positions 12 - 207 (shown in SEQ ID NO.3), for p32, select amino acids at positions 2 - 194 (shown in SEQ ID NO.4), for p22, select amino acids at positions 71 - 177 (shown in SEQ ID NO.5), for B602L, select amino acids at positions 2 - 167 (shown in SEQ ID NO.6), for CD2v, select amino acids at positions 17 - 197 (shown in SEQ ID NO.7), for E120R, select amino acids at positions 2 - 122 (shown in SEQ ID NO.8), for E199L, select amino acids at positions 2 - 155 (shown in SEQ ID NO.9), and for E248R, select amino acids at positions 77 - 191 (shown in SEQ ID NO.10). Convert the above amino acid sequences into corresponding nucleotide sequences using online software according to the codons preferred by Lactobacillus plantarum. Then, use the nucleic acid restriction endonucleases SalI and HindIII, as well as T4 DNA ligase, to clone the synthesized genes into the Lactobacillus plantarum expression vector pSlpA-LysM-DC constructed by the applicant before, which contains the constitutive promoter sequence of the Lactobacillus S-layer protein SlpA (shown in SEQ ID NO.11), the cell lysin motif (LysM) anchoring region of the extracellular transglycosylase Lp_3014 of Lactobacillus plantarum strain WFCS1 (shown in SEQ ID NO.12), and the dendritic cell-binding peptide sequence (the plasmid map is shown in Figure 1 and Figure 2 ).

[0011] S1.2: Transform the ligation products in step S1.1 into Escherichia coli DH5α competent cells. The specific operation is to add 10 μL of the ligation product to 100 μL of competent cells, immediately place on ice for 20 minutes, heat shock at 42 °C for 90 s, then add SOB liquid medium, place on a shaker at 37 °C and incubate with shaking for 45 minutes. Spread 100 μL of the culture on an LB solid medium containing 200 μg / mL of erythromycin, culture in an incubator at 37 °C for 18 hours. Pick monoclonal colonies from the plate and inoculate them into an LB liquid medium containing 200 μg / mL of erythromycin, culture on a shaker at 37 °C for 16 - 18 hours. Extract plasmids from the culture using a commercial plasmid extraction kit, and identify the plasmids by double digestion with HindIII and SalI. After double digestion with HindIII and SalI, pSlpA-LysM-DC-p72 shows 3970 bp and 642 bp; pSlpA-LysM-DC-p54 plasmid shows 3970 bp and 441 bp; pSlpA-LysM-DC-p49 shows 3970 bp and 657 bp; pSlpA-LysM-DC-p32 shows 3970 bp and 648 bp; pSlpA-LysM-DC-p22 shows 3970 bp and 390 bp; pSlpA-LysM-DC-B602L shows 3970 bp and 567 bp; pSlpA-LysM-DC-CD2v shows 3970 bp and 612 bp; pSlpA-LysM-DC-E120R shows 3970 bp and 432 bp, pSlpA-LysM-DC-E199L shows 3970 bp and 531 bp and pSlpA-LysM-DC-E248R shows 3970 bp and 414 bp( Figure 3 ), and the correctly identified transformants are named pSlpA-LysM-DC-p72, pSlpA-LysM-DC-p54, pSlpA-LysM-DC-p49, pSlpA-LysM-DC-p32, pSlpA-LysM-DC-p22, pSlpA-LysM-DC-B602L, pSlpA-LysM-DC-CD2v, pSlpA-LysM-DC-E120R, pSlpA-LysM-DC-E199L and pSlpA-LysM-DC-E248R respectively.

[0012] S2: Transformation and expression identification of recombinant Lactobacillus plantarum.

[0013] S2.1: Preparation and transformation of competent cells of Lactobacillus plantarum WFCS1: Inoculate Lactobacillus plantarum WFCS1 into MRS broth containing 1% glycine and statically culture at 37 °C until the early logarithmic phase (OD600nm is about 0.22). Centrifuge to collect cells at 1500 rpm, wash twice with ice-cold washing buffer, and resuspend in ice-cold electroporation buffer (1 M sucrose, 3 mM MgCl 2 , pH 7.4). Take 2 μg of recombinant plasmids pSlpA-LysM-DC-p72, pSlpA-LysM-DC-p54, pSlpA-LysM-DC-p49, pSlpA-LysM-DC-p32, pSlpA-LysM-DC-p22, pSlpA-LysM-DC-B602L, pSlpA-LysM-DC-CD2v, pSlpA-LysM-DC-E120R, pSlpA-LysM-DC-E199L, and pSlpA-LysM-DC-E248R, and add them to 50 μL of ice-cold competent cell suspension of Lactobacillus plantarum WFCS1 (10 9 CFU / mL) in a disposable electroporation cuvette (Gene electroporation cuvette, 0.2 cm electrode gap), and quickly use GenePulserxcell TM (BioRad, USA) to perform electroporation under the conditions of 2.0 kV, 200 Ω, and 25 μF capacitance. Immediately after the pulse, dilute the cell suspension in the electroporation cuvette to 1 mL in MRS broth and incubate at 37 °C for 2 - 3 hours. Spread the culture on MRS solid medium containing 3 μg / mL erythromycin and culture in a 37 °C incubator for 24 hours to screen for positive transformants.

[0014] S2.2: Detection of ASFV protein expression in recombinant Lactobacillus plantarum: To verify the correct expression of ASFV antigen in Lactobacillus plantarum, pick monoclonal colonies on MRS plates containing each recombinant plasmid positive transformant, inoculate them into 5 mL of MRS broth, and statically culture overnight in a 37 °C incubator. Dilute the overnight culture into 50 ml of fresh pre-warmed MRS broth containing 5 μg / ml erythromycin to make the cell density reach OD600 = 0.1, and statically culture in a 37 °C incubator for 4 hours. When OD600 = 0.6 - 0.9, centrifuge to collect cells at 4000 g for 10 minutes at 4 °C, and use PBS (137 mM NaCl, 2.7 mM KCl, 2 mM KH 2 PO 4 , 10 mM Na 2 HPO 4, cells were washed with PBS (pH 7.4), and the washed cells were resuspended in 1 ml of PBS for indirect immunofluorescence assay to verify the surface display of ASFV antigen on Lactobacillus plantarum. Another aliquot was resuspended in 1 ml of PBS containing 20 μL of 50 mM PMSF, and after sonication lysis, Western blot was performed to verify the correct expression of ASFV antigen proteins.

[0015] S2.2.1: Indirect immunofluorescence identification of recombinant Lactobacillus plantarum surface-displaying ASFV antigen: Both the ASFV antigen and the dendritic cell-binding peptide have a hexahistidine tag at their ends. Therefore, it is convenient to use a commercially available fluorescein isothiocyanate (FITC)-labeled monoclonal antibody against the histidine tag to detect whether the ASFV antigen is displayed on the surface of Lactobacillus plantarum. Take 200 μL of recombinant Lactobacillus plantarum resuspended in PBS, add a 1:200 diluted FITC-labeled monoclonal antibody against the histidine tag, incubate in the dark at room temperature for 30 minutes, wash twice with PBS, and observe the surface fluorescence of recombinant Lactobacillus plantarum under a fluorescence microscope. The results showed that all 10 recombinant Lactobacillus plantarum strains could efficiently display the ASFV antigen on the surface ( Figure 4 ).

[0016] S2.2.2: Western blot identification of recombinant Lactobacillus plantarum expressing ASFV antigen: The lysate of recombinant Lactobacillus plantarum was electrophoresed on a 15% SDS-PAGE gel, and then the proteins in the gel were electrotransferred onto a nitrocellulose (NC) membrane. The NC membrane was incubated with a horseradish peroxidase (HRP)-labeled anti-histidine tag antibody in the dark at room temperature for 1 hour, washed twice with PBS for 5 minutes each time, and then the protein bands were developed using a commercially available ECL chemiluminescent reagent and photographed with a chemiluminescence imager. The results showed that all 10 recombinant Lactobacillus plantarum strains could correctly express the ASFV antigen.

[0017] Furthermore, as a preferred embodiment of the present invention, in step S1.1, the GenBank accession number is MZ614662.

[0018] Furthermore, as a preferred embodiment of the present invention, in step S1.1, after being converted into the corresponding nucleotide sequence, it needs to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0019] Furthermore, as a preferred embodiment of the present invention, in step S1.2, after the plasmid is identified, for the clones showing the correct restriction enzyme map, sequencing identification is a necessary and crucial step.

[0020] Furthermore, as a preferred embodiment of the present invention, in step S2.1, the washing buffer is 5 mM sodium phosphate, 1 mM MgCl 2, pH 7.4.

[0021] Further, as a preferred embodiment of the present invention, in step S2.1, after incubation, the culture is spread on an MRS solid medium containing 3 μg / mL of erythromycin and cultured in an incubator at 37 °C for 24 hours to screen for positive transformants.

[0022] Further, as a preferred embodiment of the present invention, in step S2.2.1, the Lactobacillus plantarum uses the plasmid-free blank Lactobacillus plantarum WCFS1 as a negative control.

[0023] Preparation and application of a recombinant Lactobacillus plantarum oral vaccine, the method comprising the following steps:

[0024] S1: Expand the culture of 10 strains of recombinant Lactobacillus plantarum by 1000 mL each and perform viable cell counting, adjust the cell count of each recombinant strain to 10 7 CFU / mL, mix 10 strains of recombinant Lactobacillus plantarum equally, which is the Lactobacillus plantarum oral vaccine. Divide 100 non-diseased fattening pigs from a certain African swine fever virus (ASFV)-positive pig farm into an immunization group and a control group on average. The pigs in the immunization group are fed once every 3 days for a total of two times, and each pig is fed 10 mL of recombinant Lactobacillus plantarum through drinking water each time. The control group is fed the same amount of blank Lactobacillus plantarum WCFS1 in the same way. The results show that in the ASFV-positive pig farm, among the 50 non-diseased pigs fed with the recombinant Lactobacillus plantarum displaying ASFV antigen on the surface, only 2 showed clinical symptoms and finally died within a one-month test period, while among the 50 pigs fed with the same amount of blank Lactobacillus plantarum WCFS1, they became ill successively throughout the test period and a total of 38 died at the end of the test. This result indicates that oral administration of the recombinant Lactobacillus plantarum displaying ASFV antigen on the surface can provide effective mucosal immunity and prevent immunized pigs from being infected with ASFV in the natural environment.

[0025] Further, as a preferred embodiment of the present invention, in step S1, the oral vaccine is composed of an equal mixture of recombinant Lactobacillus plantarum strains that respectively display African swine fever virus p72, p54, p49, p32, p22, B602L, CD2v, E120R, E199L, and E248R proteins on the surface.

[0026] Further, as a preferred embodiment of the present invention, in step S1, observe the health status of the pig group daily after feeding and record the number of African swine fever-infected pigs in each group, and continuously observe for 4 weeks.

[0027] Advantages: The technical solution of this application has the following technical effects: The present invention has the advantages of not requiring an inducer, efficiently expressing antigen proteins, fusion expression enhancing immune cell uptake and processing, simple oral immunization method, stimulating mucosal immunity, and effectively preventing and controlling ASFV infection. Using Lactobacillus plantarum, a probiotic that naturally exists in the animal intestine and can colonize and replicate in the digestive tract for a long time, as a vector, the key antigenic epitopes of proteins such as nucleocapsid protein p72, outer envelope protein CD2v, inner envelope proteins p54, p22, pE199L, and pE248R, which play important roles in the adsorption, internalization, endocytosis, and membrane fusion of ASFV, are fused with dendritic cell-binding peptides and then displayed on the surface of Lactobacillus plantarum to prepare a recombinant Lactobacillus plantarum similar to the structure of ASFV. To overcome the problem that an inducer needs to be added for in vitro culture, while the inducer cannot be added when the engineered bacteria enter the body, this application uses the promoter of the constitutive expression gene of Lactobacillus plantarum to ensure that the recombinant engineered bacteria can still continuously express the antigens of ASFV after entering the animal digestive tract. Using the recombinant Lactobacillus plantarum with the above advantages as an oral vaccine can induce mucosal immunity in pigs to prevent and control ASFV infection through the digestive tract mucosa, and meet the urgent need of the pig industry to obtain long-term immunity with fewer feeding times;

[0028] Secondly, a recombinant Lactobacillus plantarum displaying 10 antigen proteins of ASFV on the surface was constructed for the first time. The engineered strain can efficiently express antigen proteins without the addition of an inducer in in vitro culture; the antigenic epitopes of 10 proteins of ASFV are fused and expressed with dendritic cell-binding peptides, ensuring the effective uptake and processing of antigens by the body's immune cells; the oral vaccine of Lactobacillus plantarum displaying ASFV antigens on the surface has a simple immunization method, which can be easily implemented by drinking water or mixing with feed, facilitating large-scale use, saving time and effort, and at the same time avoiding the stress caused by the traditional intramuscular injection immunization method; the recombinant Lactobacillus plantarum displaying 10 antigen proteins of ASFV can effectively colonize and continuously replicate after oral administration into the digestive tract, thereby stimulating the body to produce mucosal immunity against ASFV. After being used in the field, it can effectively prevent and control ASFV infection. Brief Description of the Drawings

[0029] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention and do not constitute a limitation to the present invention.

[0030] In the drawings:

[0031] Figure 1 is the recombinant plasmid map of the present invention;

[0032] Figure 2 is the restriction endonuclease HindIII + SalI digestion map of the recombinant plasmid of the present invention;

[0033] Figure 3 This is the indirect immunofluorescence identification map of the recombinant Lactobacillus plantarum surface-displaying ASFV antigen of the present invention;

[0034] Figure 4 This is the Western blot identification map of the recombinant Lactobacillus plantarum expressing ASFV antigen of the present invention. Detailed implementation manners

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. To better understand the technical content of the present invention, specific embodiments are specifically cited and described in conjunction with the accompanying drawings as follows. In the present disclosure, aspects of the present invention are described with reference to the accompanying drawings, and many illustrative embodiments are shown in the drawings. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0036] Appendix Figure 2 Among them: Supplementary description of the restriction endonuclease HindIII + SalI digestion map of the recombinant plasmid: MW: 1kb DNA Ladder;

[0037] Lane 1: pSlpA-LysM-DC-p72;

[0038] Lane 2: pSlpA-LysM-DC-p54;

[0039] Lane 3: pSlpA-LysM-DC-p49;

[0040] Lane 4: pSlpA-LysM-DC-p32;

[0041] Lane 5: pSlpA-LysM-DC-p22;

[0042] Lane 6: pSlpA-LysM-DC-B602L;

[0043] Lane 7: pSlpA-LysM-DC-CD2v;

[0044] Lane 8: pSlpA-LysM-DC-E120R;

[0045] Lane 9: pSlpA-LysM-DC-E199L;

[0046] Lane 10: pSlpA-LysM-DC-E248R.

[0047] SEQ ID NO.1 (p72 amino acid sequence 136aa - 326aa)

[0048] TFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQPP

[0049] SEQ ID NO.2 (p54 amino acid sequence 61aa - 184aa)

[0050] AIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSL

[0051] SEQ ID NO.3 (p49 amino acid sequence 12aa - 207aa)

[0052] DYRSDPPLWESDLPRHNRYSDNILNSRYCGNKNGAAPVYNEYTNSPEKAEKGLQLSDLRNFSFMLNPQHKNIGYGDAQDLEPYSSIPKNKLFNHFKNHRPAFSTHTENLIRRNVVRTEKKTFPQVASLKGTQKNCLTQPSSLPSLKNPKNSSVPSTRFSEHTKFFSYEDLPKLRTKGTIKHEQHLGDQMPGQHYNG

[0053] SEQ ID NO.4 (p32 amino acid sequence 2aa - 194aa)

[0054] DFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKK

[0055] SEQ ID NO.5 (Amino acid sequence of p22, 71aa - 177aa)

[0056] NIKIDSKISSCEFTPNFYRFTDTAADEQQEFGKTRHPIKITPSPSESHSPQEVCEKYCSWGTDDCTGWEYVGDEKEGTCYVYNNPHHPVLKYGKDHIIALPRNHKHA

[0057] SEQ ID NO.6 (Amino acid sequence of pB602L, 2aa - 167aa)

[0058] AEFNIDELLKNVLEDPSTEISEETLKQLYQRTNPYKQFKNDSRVAFCSFTNLREQYIRRLIMTSFIGYVFKALQEWMPSYSKPTHTTKTLLSELITLVDTLKQETNDVPSESVVNTILSIADSCKTQTQKSKEAKTTIDSFLREHFVFDPNLHAQSAYTCADTNVD

[0059] SEQ ID NO.7 (Amino acid sequence of pCD2v, 17aa - 197aa)

[0060] DYWVSFNKTIILDSNITNDNNDINGVSWNFFNNSFNTLATCGKAGNFCECSNYSTSIYNITNNCSLTIFPHNDVFDTTYQVVWNQIINYTIKLLTPATPPNITYNCTNFLITCKKNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFTATCIINNTISTSNETTLINCTYLTLSSN

[0061] SEQ ID NO.8 (Amino acid sequence of pE120R, 2aa - 122aa)

[0062] ADFNSPIQYLKEDSRDRTSIGSLEYDENADTMIPSFAAGLEEFEPIPDYDPTTSTSLYSQLTHNMEKIAEEEDSNFLHDTREFTSLVPDEADNKPEDDEESGAKPKKKKHLFPKLSSHKSK

[0063] SEQ ID NO.9 (Amino acid sequence of pE199L, 2aa - 155aa)

[0064] SCMPVSTKCNDIWVDFSCTGPSISELQKKEPKAWAAILRSHTNQQTAEDDNIIGSICDKQGLCSKDEYAYSQYCACVNSGTLWAECAFAPCNGNKNAYKTTEQRNILTNKQCPSGLTICQNIAEYGGSGNISDLYQNFNCNSVINTFLINVMNH

[0065] SEQ ID NO.10 (Amino acid sequence of pE248R, 77aa - 191aa)

[0066] SNQITQNLKDQEVALTQWMDAGTHDQKTDIEENIKVNLTTTLIQNCVSSLSGMNVLVVKGNGNIVENATQKQSQQIISNCLQGSKQAIDTTTGITNTVNQYSHYTSKNFFDFIAD

[0067] SEQ ID NO.11 (SlpA promoter sequence)

[0068] GCCAATAGAAAAAGCGAACCTAATAAGATTAATCTTTAGGAAAATCGAATAAAAATATTACTTTTTTGATATGTTTTGTCATAGTTTCGTAAAATTTAGTAAAGATTACGAGCGATAAATAGAGAACTTAATCTTGTCTTTTTCTTGCTATAGCTAGGTTTAGCACATTTTACAATTTTAAAGTGCTTGTAATGCTTGTGGGGGTAAGCGGTAGGTGAAATATTACAAATAGTATTTTTCGGTCATTTTAACTTGCTATTTCTTGAAGAGGTTAGTACAATATGAATCGTGGTAAGTAATAGGACGTGCTTCAGGCGTGTTGCCTGTACGCATGCTGATTCTTCAGCAAGACTACTACCTCATGAGAGTTATAGACTCATGGATCTTGCTTTGAAGGGTTTTGTACATTATAGGCTCCTATCACATGCTGAACCTATGGCCTATTACATTTTTTTATATTTCAAGGAGGAAAAGACCA

[0069] SEQ ID NO.12 (LysM sequence)

[0070] ATGAAAAAACTTGTAAGTACAATCGTAACTACCTCAGCTGCAGCCGCTGGTTTATTATTCGCCGGTGTCCTCAATGCTAACGCCGACTCAACTTACACCGTTAAGAGCGGTGATTCCGTTTGGGCCATCGCACAAAAATTCAACACAACTATCAATCATGTTGAAACGACTAATAACATCAAGGGTCACTACATCTTACCTGGTCAAAAACTATCTATTAAGACGAGTTCAACCAGTTCTGATAACAACACCTCATCAACGACTTCAAACACAACGAGTTCAAGTGCTTCAACGACTAGTTCTAGTTCAACTAGCAACACAACCGCGACGACCACTTCAACGAGTTCAACTGACTCCACGACTTCAAGCTACACTGGTAGCAATTTGAAGAGTTACGTTTTAAGTCAGATGCAATCACGGACTGGTGTCTCAGCTTCGACTTGGAACACGATTATTACGCGTGAATCCAACTGGCAACCATACGTCCGCAACAGTTCTAGTGGTGCCTACGGGTTATTCCAGAACATGCACATCAGCAGTGGTTCTGTTGAAGAACAAGTTAACGCTGCCGTTGCTTTATATGAAGCACAAGGTATGGCTGCTTGGGCCCTT

[0071] SEQ ID NO.13 (Dendritic cell-binding peptide sequence)

[0072] TTTTATCCAAGTTATCATAGTACGCCACAACGGCCA

[0073] As attached Figure 1 To attached Figure 4 As shown: This example provides the construction of recombinant Lactobacillus plantarum of African swine fever virus antigen, and its method includes the following steps:

[0074] S1: Construction of Lactobacillus plantarum shuttle vectors containing different antigen genes of ASFV.

[0075] S1.1: Refer to the genomic sequence of the ASFV Chinese isolate CADC_HN09 published in GenBank to obtain the protein sequences of p72, p54, p49, p32, p22, B602L, CD2v, E120R, E199L, and E248R respectively. Use the Protean program in the Lasergene software package to analyze the antigenic site concentration regions of each protein. For p72, select amino acids at positions 136 - 326 (shown in SEQ ID NO.1), for p54, select amino acids at positions 61 - 184 (shown in SEQ ID NO.2), for p49, select amino acids at positions 12 - 207 (shown in SEQ ID NO.3), for p32, select amino acids at positions 2 - 194 (shown in SEQ ID NO.4), for p22, select amino acids at positions 71 - 177 (shown in SEQ ID NO.5), for B602L, select amino acids at positions 2 - 167 (shown in SEQ ID NO.6), for CD2v, select amino acids at positions 17 - 197 (shown in SEQ ID NO.7), for E120R, select amino acids at positions 2 - 122 (shown in SEQ ID NO.8), for E199L, select amino acids at positions 2 - 155 (shown in SEQ ID NO.9), and for E248R, select amino acids at positions 77 - 191 (shown in SEQ ID NO.10). Use online software to convert the above amino acid sequences into corresponding nucleotide sequences according to the codons preferred by Lactobacillus plantarum, and use the nucleic acid restriction endonucleases SalI and HindIII, as well as T4 DNA ligase, to clone the synthesized gene into the Lactobacillus expression vector pSlpA-LysM-DC containing the constitutive promoter sequence of Lactobacillus S-layer protein SlpA (shown in SEQ ID NO.11), the cell lysin motif (LysM) anchoring region of the extracellular transglycosylase Lp_3014 of Lactobacillus plantarum WFCS1 strain (shown in SEQ ID NO.12), and the dendritic cell-binding peptide sequence (the plasmid map is shown in Figure 1 and Figure 2 ).

[0076] Furthermore, in step S1.1, the GenBank accession number is MZ614662.

[0077] Moreover, in step S1.1, after converting into the corresponding nucleotide sequences, they need to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0078] S1.2: Transform the ligation products in step S1.1 into competent Escherichia coli DH5α cells. The specific operation is to add 10 μL of the ligation product to 100 μL of competent cells, immediately ice-bath for 20 minutes, heat-shock at 42 °C for 90 s, then add SOB liquid medium, place it on a shaker at 37 °C and shake culture for 45 minutes. Spread 100 μL of the culture on an LB solid medium containing 200 μg / mL of erythromycin, culture it in an incubator at 37 °C for 18 hours. Pick monoclonal colonies from the plate and inoculate them into an LB liquid medium containing 200 μg / mL of erythromycin, culture them on a shaker at 37 °C for 16 - 18 hours. Extract plasmids from the culture using a commercial plasmid extraction kit, and identify the plasmids by double digestion with HindIII and SalI. After double digestion with HindIII and SalI, pSlpA-LysM-DC-p72 shows 3970 bp and 642 bp; pSlpA-LysM-DC-p54 plasmid shows 3970 bp and 441 bp; pSlpA-LysM-DC-p49 shows 3970 bp and 657 bp; pSlpA-LysM-DC-p32 shows 3970 bp and 648 bp; pSlpA-LysM-DC-p22 shows 3970 bp and 390 bp; pSlpA-LysM-DC-B602L shows 3970 bp and 567 bp; pSlpA-LysM-DC-CD2v shows 3970 bp and 612 bp; pSlpA-LysM-DC-E120R shows 3970 bp and 432 bp, pSlpA-LysM-DC-E199L shows 3970 bp and 531 bp and pSlpA-LysM-DC-E248R shows 3970 bp and 414 bp( Figure 3 ), and the correctly identified transformants are named pSlpA-LysM-DC-p72, pSlpA-LysM-DC-p54, pSlpA-LysM-DC-p49, pSlpA-LysM-DC-p32, pSlpA-LysM-DC-p22, pSlpA-LysM-DC-B602L, pSlpA-LysM-DC-CD2v, pSlpA-LysM-DC-E120R, pSlpA-LysM-DC-E199L and pSlpA-LysM-DC-E248R respectively.

[0079] Furthermore, in step S1.2, after identifying the plasmids, for the clones presenting the correct restriction enzyme maps, sequencing identification is a necessary and crucial step.

[0080] S2: Transformation and expression identification of recombinant Lactobacillus plantarum.

[0081] S2.1: Preparation and transformation of competent cells of Lactobacillus plantarum WFCS1: Inoculate Lactobacillus plantarum WFCS1 into MRS broth containing 1% glycine, and statically culture at 37 °C until the early logarithmic phase (OD600nm is about 0.22). Collect the cells by centrifugation at 1500 rpm, wash twice with ice-cold washing buffer, and resuspend in ice-cold electroporation buffer (1 M sucrose, 3 mM MgCl 2 , pH 7.4). Take 2 μg of recombinant plasmids pSlpA-LysM-DC-p72, pSlpA-LysM-DC-p54, pSlpA-LysM-DC-p49, pSlpA-LysM-DC-p32, pSlpA-LysM-DC-p22, pSlpA-LysM-DC-B602L, pSlpA-LysM-DC-CD2v, pSlpA-LysM-DC-E120R, pSlpA-LysM-DC-E199L, and pSlpA-LysM-DC-E248R, and add them separately to disposable electroporation cuvettes (Gene electroporation cuvette, 0.2 cm electrode gap) containing 50 μL of ice-cold competent cell suspension of Lactobacillus plantarum WFCS1 (109 CFU / mL). Immediately use GenePulserxcell TM (BioRad, USA) to perform electroporation under the conditions of 2.0 kV, 200 Ω, and a capacitance of 25 μF. Immediately after the pulse, dilute the cell suspension in the electroporation cuvette to 1 mL in MRS broth and incubate at 37 °C for 2 - 3 hours. Spread the culture on MRS solid medium containing 3 μg / mL of erythromycin and culture in a 37 °C incubator for 24 hours to screen for positive transformants.

[0082] Furthermore, in step S2.1, the washing buffer is 5 mM sodium phosphate, 1 mM MgCl 2 , pH 7.4.

[0083] Moreover, in step S2.1, after incubation, spread the culture on MRS solid medium containing 3 μg / mL of erythromycin and culture in a 37 °C incubator for 24 hours to screen for positive transformants.

[0084] S2.2: Detection of ASFV protein expressed by recombinant Lactobacillus plantarum: To verify the correct expression of ASFV antigen by Lactobacillus plantarum, single colonies were picked from MRS plates containing each positive transformant of the recombinant plasmid and inoculated into 5 mL of MRS broth. The cultures were incubated overnight at 37 °C in a static incubator. The overnight cultures were diluted into 50 mL of fresh pre-warmed MRS broth containing 5 μg / mL erythromycin to a cell density of OD600 = 0.1, and incubated at 37 °C in a static incubator for 4 hours. When OD600 = 0.6 - 0.9, the cells were harvested by centrifugation at 4000 g for 10 minutes at 4 °C and washed with PBS (137 mM NaCl, 2.7 mM KCl, 2 mM KH 2 PO 4 、10 mM Na 2 HPO 4 , pH 7.4). The washed cells were resuspended in 1 mL of PBS for indirect immunofluorescence assay to verify the surface display of ASFV antigen on Lactobacillus plantarum. Another aliquot was resuspended in 1 mL of PBS containing 20 μL of 50 mM PMSF and sonicated for Western blot to verify the correct expression of ASFV antigen proteins.

[0085] S2.2.1: Indirect immunofluorescence identification of ASFV antigen surface-displayed on recombinant Lactobacillus plantarum: Both the ASFV antigen and the dendritic cell-binding peptide are tagged with a hexahistidine tag at the end, so it is convenient to use a commercially available fluorescein isothiocyanate (FITC)-labeled anti-histidine tag monoclonal antibody to detect whether the ASFV antigen is surface-displayed on Lactobacillus plantarum. Take 200 μL of recombinant Lactobacillus plantarum resuspended in PBS, add the FITC-labeled anti-histidine tag monoclonal antibody diluted 1:200, and incubate in the dark at room temperature for 30 minutes. Wash twice with PBS and observe the surface fluorescence of recombinant Lactobacillus plantarum under a fluorescence microscope. The results showed that all 10 recombinant Lactobacillus plantarum strains could efficiently surface-display the ASFV antigen ( Figure 4 ).

[0086] Furthermore, in step S2.2.1, the plasmid-free blank Lactobacillus plantarum WCFS1 was used as a negative control for Lactobacillus plantarum.

[0087] S2.2.2: Western blot identification of recombinant Lactobacillus plantarum expressing ASFV antigen: The lysate of recombinant Lactobacillus plantarum was electrophoresed on a 15% SDS-PAGE gel, and then the proteins in the gel were electrotransferred onto a nitrocellulose (NC) membrane. The NC membrane was incubated with a histidine-tag antibody labeled with horseradish peroxidase (HRP) in the dark at room temperature for 1 hour, washed twice with PBS for 5 minutes each time, and then the protein bands were developed using a commercial ECL chemiluminescent reagent and photographed with a chemiluminescent imager. The results showed that all 10 recombinant Lactobacillus plantarum strains could correctly express the ASFV antigen.

[0088] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations.

[0089] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention pertains can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the scope defined in the claims.

Claims

1. Construction of recombinant Lactobacillus plantarum of African swine fever virus antigen, characterized in that: The method comprises the following steps: S1: Construction of Lactobacillus plantarum shuttle vectors containing genes for different ASFV antigens; S1.1: With reference to the genome sequence of the ASFV Chinese isolate CADC_HN09 published in GenBank, the p72, p54, p49, p32, p22, B602L, CD2v, E120R, E199L and E248R protein sequences were obtained respectively, and the Protean program in the Lasergene software package was used to analyze the concentrated antigenic sites of each protein, among which amino acids 136-326 were selected for p72, 61-184 for p54, 12-207 for p49, and 136-326 for p72. amino acids, p32 selected amino acids 2-194, p22 selected amino acids 71-177, B602L selected amino acids 2-167, CD2v selected amino acids 17-197, E120R selected amino acids 2-122, E199L selected amino acids 2-155 and E248R selected amino acids 77-191, the above amino acid sequences were converted into corresponding nucleotide sequences according to the codons preferred by Lactobacillus plantarum using online software, and the synthesized genes were cloned into the constitutive promoter sequence of the lactobacillus S layer protein SlpA, the lysin motif (LysM) anchor region of the extracellular transglycosylase Lp_3014 of the Lactobacillus plantarum WFCS1 strain, and the dendritic cell binding peptide sequence constructed by the applicant using nucleic acid restriction endonucleases SalI and HindIII, and T4 DNA ligase. S1.2: The ligation products in step S1.1 were transformed into E. coli DH5α competent cells. The specific operation was to add 10 μL of the ligation product to 100 μL of competent cells, immediately ice-bath for 20 minutes, heat-shock at 42°C for 90 seconds, then add SOB liquid culture medium, place in a 37°C shaker for 45 minutes, spread 100 μL of the culture on LB solid culture medium containing 200 μg / mL erythromycin, and culture in a 37°C incubator for 18 hours. Single clones were picked from the plate. The cells were inoculated into LB liquid culture medium containing 200 μg / mL erythromycin and cultured at 37°C in a shaking incubator for 16-18 hours. Plasmids were extracted from the culture using a commercial plasmid extraction kit and identified using HindIII and SalI double digestion. After HindIII and SalI double digestion, pSlpA-LysM-DC-p72 showed 3970 bp and 642 bp; pSlpA-LysM-DC-p54 plasmids showed 3970 bp and 441 bp. pSlpA-LysM-DC-p49 presented 3970 bp and 657 bp; pSlpA-LysM-DC-p32 showed 3970bp and 648bp; pSlpA-LysM-DC-p22 showed 3970bp and 390bp; pSlpA-LysM-DC-B602L showed 3970bp and 567bp; pSlpA-LysM-DC-CD2v showed 3970bp and 612bp; pSlpA-LysM-DC-E120R showed 3970bp and 432bp, pSlpA-LysM-DC-E199L showed 3970bp and 531bp and pSlpA-LysM-DC-E248 showed R presented 3970 bp and 414 bp, and the correct transformants were identified and named pSlpA-LysM-DC-p72, pSlpA-LysM-DC-p54, pSlpA-LysM-DC-p49, pSlpA-LysM-DC-p32, pSlpA-LysM-DC-p22, pSlpA-LysM-DC-B602L, pSlpA-LysM-DC-CD2v, pSlpA-LysM-DC-E120R, pSlpA-LysM-DC-E199L, and pSlpA-LysM-DC-E248R, respectively; S2: Transformation and expression identification of recombinant Lactobacillus plantarum; S2.1: Preparation and transformation of competent cells of Lactobacillus plantarum WFCS1: Lactobacillus plantarum WFCS1 was inoculated into MRS broth containing 1% glycine and cultured at 37°C until it reached the early logarithmic phase (OD 600nm about 0.22). The cells were collected by centrifugation at 1500rpm, washed twice with ice-cold washing buffer, and resuspended in ice-cold electroporation buffer (1M sucrose, 3mM MgCl2, pH7.4). 2 μg of recombinant plasmids pSlpA-LysM-DC-p72, pSlpA-LysM-DC-p54, pSlpA-LysM-DC-p49, pSlpA-LysM-DC-p32 were taken. , pSlpA-LysM-DC-p22, pSlpA-LysM-DC-B602L, pSlpA-LysM-DC-CD2v, pSlpA-LysM-DC-E120R, pSlpA-LysM-DC-E199L and pSlpA-LysM-DC-E248R were added to 50 μL ice-cold Lactobacillus plantarum WFCS1 competent cell suspension (10 9 CFU / mL) in a disposable electroporation cup (Gene Electroporation cup, 0.2 cm electrode gap), quickly use GenePulser xcell TM (BioRad, USA) was electroporated under the conditions of 2.0 kV, 200 Ω and 25 μF capacitance. After the pulse, the cell suspension in the electroporation cup was quickly diluted to 1 mL in MRS broth and incubated at 37 °C for 2-3 h. The culture was spread on MRS solid medium containing 3 μg / mL erythromycin and cultured in a 37 °C incubator for 24 h to screen positive transformants; S2.2: Detection of ASFV protein expressed in recombinant Lactobacillus plantarum: In order to verify the correct expression of ASFV antigens by Lactobacillus plantarum, a single clone was picked from the MRS plate containing each recombinant plasmid positive transformant and inoculated into 5 mL of MRS broth. The culture was incubated overnight at 37°C. The overnight culture was diluted into 50 ml of fresh preheated MRS broth containing 5 μg / ml erythromycin to a cell density of OD600 = 0.1 and incubated at 37°C for 4 hours. When OD600 = 0.6-0.9, the cells were collected by centrifugation at 4000 g for 10 minutes at 4°C and washed with PBS (137 mM NaCl, 2.7 mM KCl, 2 mM KH2PO4, 10 mM Na2HPO4, pH 7.4). The washed cells were resuspended in 1 ml of PBS for indirect immunofluorescence test to verify that the ASFV antigens were displayed on the surface of Lactobacillus plantarum. Another portion was resuspended in 1 ml of 20 μL In PBS containing 50 mM PMSF, after ultrasonic lysis, Western blot was performed to verify the correct expression of each antigen protein of ASFV; S2.2.1: Indirect immunofluorescence identification of ASFV antigens displayed on the surface of recombinant Lactobacillus plantarum: The ends of the ASFV antigen and the dendritic cell binding peptide are both labeled with hexahistidine tags. Therefore, it is convenient to use commercial fluorescein isothiocyanate (FITC)-labeled anti-histidine tag monoclonal antibodies to detect whether the ASFV antigen is displayed on the surface of Lactobacillus plantarum. Take 200 μL of PBS-resuspended recombinant Lactobacillus plantarum, add 1:200 diluted FITC-labeled anti-histidine tag monoclonal antibodies, incubate at room temperature in the dark for 30 minutes, wash twice with PBS, and observe the surface fluorescence of recombinant Lactobacillus plantarum under a fluorescence microscope. The results showed that all 10 recombinant Lactobacillus plantarum can efficiently display ASFV antigens on the surface; S2.2.2: Western blot identification of ASFV antigen expressed by recombinant Lactobacillus plantarum: The lysate of recombinant Lactobacillus plantarum was electrophoresed in a 15% SDS-PAGE gel, and the proteins in the gel were then electroblotted onto a nitrocellulose (NC) membrane. The NC membrane was incubated with an anti-histidine tag antibody labeled with horseradish peroxidase (HRP) at room temperature in the dark for 1 hour, washed twice with PBS for 5 minutes each, and then the protein bands were developed using a commercial ECL chemiluminescent reagent and photographed with a chemiluminescent imager. The results showed that all 10 recombinant Lactobacillus plantarum could correctly express the ASFV antigen.

2. Preparation and application of recombinant Lactobacillus plantarum oral vaccine, characterized in that: The method comprises the following steps: S1: Expand the culture of 10 recombinant Lactobacillus plantarum strains, 1000 mL each, and count the viable bacteria. Adjust the number of each recombinant strain to 10 7 CFU / mL, 10 strains of recombinant Lactobacillus plantarum were mixed in equal amounts to obtain the Lactobacillus plantarum oral vaccine, and 100 healthy fattening pigs from an African swine fever virus-positive pig farm were evenly divided into an immunization group and a control group. The pigs in the immunization group were fed once every 3 days, for a total of two times, and each pig was fed 10mL of recombinant Lactobacillus plantarum through drinking water each time. The control group was fed with an equal amount of blank Lactobacillus plantarum WCFS1 in the same way. The results showed that in an ASFV-positive pig farm, only 2 of the 50 healthy pigs fed with recombinant Lactobacillus plantarum displaying ASFV antigens on the surface showed clinical symptoms during the one-month trial period and eventually died, while the 50 pigs fed with an equal amount of blank Lactobacillus plantarum WCFS1 became ill one after another throughout the trial period, and 38 pigs died at the end of the trial. This result shows that oral administration of recombinant Lactobacillus plantarum displaying ASFV antigens on the surface can provide effective mucosal immunity and prevent immunized pigs from being infected with ASFV in the natural environment.

3. The construction of the recombinant Lactobacillus plantarum of African swine fever virus antigen according to claim 1, characterized in that: In the step S1.1, GenBank accession number is MZ614662.

4. The construction of the recombinant Lactobacillus plantarum of African swine fever virus antigen according to claim 1, characterized in that: In the step S1.1, after conversion into the corresponding nucleotide sequence, it needs to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

5. The construction of the recombinant Lactobacillus plantarum of African swine fever virus antigen according to claim 1, characterized in that: In step S1.2, after the plasmid is identified, sequencing identification is a necessary and critical step for clones that present the correct restriction endonuclease map.

6. The construction of the recombinant Lactobacillus plantarum of African swine fever virus antigen according to claim 1, characterized in that: In step S2.1, the washing buffer is 5 mM sodium phosphate, 1 mM MgCl2, pH 7.

4.

7. The construction of the recombinant Lactobacillus plantarum of African swine fever virus antigen according to claim 1, characterized in that: In the step S2.1, after incubation, the culture was spread on MRS solid medium containing 3 μg / mL erythromycin, cultured in a 37° C. incubator for 24 hours, and positive transformants were screened.

8. The construction of the recombinant Lactobacillus plantarum of African swine fever virus antigen according to claim 1, characterized in that: In the step S2.2.1, the blank Lactobacillus plantarum WCFS1 without plasmid is used as a negative control.

9. The preparation and immune efficacy evaluation of the recombinant Lactobacillus plantarum oral vaccine according to claim 2, characterized in that: In step S1, the oral vaccine is prepared by mixing equal amounts of recombinant Lactobacillus plantarum that display African swine fever virus p72, p54, p49, p32, p22, B602L, CD2v, E120R, E199L and E248R proteins on their surfaces.

10. The preparation and immune efficacy evaluation of the recombinant Lactobacillus plantarum oral vaccine according to claim 2, characterized in that: In step S1, the health status of the pigs is observed every day after feeding, and the number of pigs infected with African swine fever in each group is recorded for four consecutive weeks.

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